Intracellular Ca2+ signaling via changes in cytosolic Ca2+ concentration controls a wide range of cellular and physiologic processes. Ca2+ mobilization from intracellular stores mediated by second messengers is an important source of cytosolic Ca2+. Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent Ca2+-mobilizing second messenger identified to date; it uniquely mobilizes Ca2+ from acidic endolysosomal organelles. NAADP has been shown to be effective in evoking Ca2+ release in a multitude of different mammalian cells and defects in NAADP signaling are now being implicated in many diseases. Despite the importance of NAADP-evoked Ca2+ signaling, the molecular identities of the NAADP receptors and Ca2+- release channels involved in this process have yet to be unequivocally defined. Accumulated evidence indicates that endolysosomal two-pore channels (TPCs) might participate in NAADP-evoked Ca2+ release. However, recent direct patch-clamp recordings of TPC channel currents in endolysosomes showed that TPCs were Na+-selective channels with very limited Ca2+ permeability, and the channel activity was not sensitive to NAADP. Currently, a unifying hypothesis about the role of TPCs in NAADP signaling is that TPCs are not directly involved in NAADP binding but are part of the NAADP receptor/Ca2+-release channel complex, in which the molecular identities of other key proteins remain unknown. These new findings and speculations warrant an exploration of additional proteins that may be important for NAADP-evoked Ca2+ signaling. In the proposed study, we will use both TPCs and NAADP as baits to isolate their interacting partners from mammalian HEK293 and SKBR3 cells and then use unbiased quantitative proteomic analysis and functional assays to screen and identify novel proteins important for NAADP-evoked Ca2+ signaling. We will pursue the following two specific aims. Specific Aim 1. Identify novel TPC-interacting proteins important for NAADP-evoked Ca2+ signaling. We aim to systematically determine the interactome of TPCs using a SILAC-based quantitative proteomic approach with advanced mass spectrometry. We will then perform Ca2+ imaging functional assays on identified TPC-interacting proteins to identify novel proteins important for NAADP-evoked Ca2+ signaling. Specific Aim 2. Identify NAADP-interacting proteins important for NAADP-evoked Ca2+ signaling. To directly target the NAADP receptor, we will perform affinity purification of NAADP-interacting proteins from HEK293 and SKBR3 cells using immobilized NAADP and its analogue and then employ quantitative proteomic analysis and functional assays to identify novel proteins important for NAADP-induced Ca2+ release. The proposed studies are designed to use systematic approaches to unbiasedly screen and identify novel proteins important for NAADP-evoked Ca2+ release. Our results will likely generate a breakthrough in the molecular basis and mechanisms of NAADP signaling by identifying novel proteins that could serve as NAADP receptors, Ca2+- release channels, or regulatory proteins necessary for NAADP-evoked Ca2+ release. 1